The present invention relates to therapeutic CNTF-related polypeptides useful for the treatment of neurological or other diseases or disorders.
Ciliary neurotrophic factor (CNTF) is a protein that is required for the survival of embryonic chick ciliary ganglion neurons in vitro (Manthorpe et al., 1980, J. Neurochem. 34:69-75). The ciliary ganglion is anatomically located within the orbital cavity, lying between the lateral rectus and the sheath of the optic nerve; it receives parasympathetic nerve fibers from the oculomotor nerve which innervates the ciliary muscle and sphincter pupillae.
Over the past decade, a number of biological effects have been ascribed to CNTF in addition to its ability to support the survival of ciliary ganglion neurons. CNTF is believed to induce the differentiation of bipotential glial progenitor cells in the perinatal rat optic nerve and brain (Hughes et al., 1988, Nature 335:70-73). Furthermore, it has been observed to promote the survival of embryonic chick dorsal root ganglion sensory neurons (Skaper and Varon, 1986, Brain Res. 389:39-46). In addition, CNTF supports the survival and differentiation of motor neurons, hippocampal neurons and presympathetic spinal cord neurons [Sendtner, et al., 1990, Nature 345: 440-441; Ip, et al. 1991, J. Neurosci. 11:3124-3134; Blottner, et al. 1989, Neurosci. Lett. 105:316-320].
It has long been known that innervation of skeletal muscle plays a critical role in the maintenance of muscle structure and function. Skeletal muscle has been shown recently to be a target of positive CNTF actions. Specifically, CNTF prevents both the denervation-induced atrophy (decreased wet weight and myofiber cross sectional area) of skeletal muscle and the reduced twitch and tetanic tensions of denervated skeletal muscle. Helgren et al., 1994, Cell 76:493-504. In this model, human CNTF also produces an adverse effect that is manifested as a retardation of weight gain. This adverse effect has also been observed in clinical trials with rHCNTF for the treatment of ALS. Therefore, simultaneous measurements of muscle weight and animal body weight following denervation could be used as a measure of efficacy and adverse reaction, respectively, in response to treatment with rHCNTF or other compounds. The ratio of the potency values obtained from these measurements is defined as the therapeutic index (T.I.), expressed here as TD25/ED50, so that the higher the value of T.I., the safer the compound at a therapeutic dose.
CNTF has been cloned and synthesized in bacterial expression systems, as described by Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and in International Publication No. WO 91/04316, published on Apr. 4, 1991, which are incorporated by reference in their entirety herein.
The receptor for CNTF (termed xe2x80x9cCNTFRxcex1xe2x80x9d) has been cloned, sequenced and expressed [see Davis, et al., 1991 Science 253:59-63]. CNTF and the hemopoietic factor known as leukemia inhibitory factor (LIF) act on neuronal cells via a shared signaling pathway that involves the IL-6 signal transducing component gp130 as well as a second, xcex2-component (know as LIFR xcex2); accordingly, the CNTF/CNTF receptor complex can initiate signal transduction in LIF responsive cells, or other cells which carry the gp130 and LIFRxcex2 components [Ip, et al., 1992, Cell 69:1121-1132].
In addition to human CNTF, the corresponding rat (Stxc3x6ckli et al., 1989, Nature 342:920-923), and rabbit (Lin et al., 1989, J. Biol. Chem. 265:8942-8947) genes have been cloned and found to encode a protein of 200 amino acids, which share about 80% sequence identity with the human gene. Both the human and rat recombinant proteins have been expressed at exceptionally high levels (up to 70% of total protein) and purified to near homogeneity.
Despite their structural and functional similarity, recombinant human and rat CNTF differ in several respects. The biological activity of recombinant rat CNTF in supporting survival and neurite outgrowth from embryonic chick ciliary neurons in culture is four times better than that of recombinant human CNTF [Masiakowski et al., 1991, J. Neurochem. 57:1003-1012]. Further, rat CNTF has a higher affinity for the human CNTF receptor than does human CNTF.
A surprising difference in the physical properties of human and rat CNTF, which are identical in size, is their different mobility on SDS gels. This difference in behavior suggests the presence of an unusual structural feature in one of the two molecules that persists even in the denatured state (Masiakowski et al., 1991, J. Neurochem. 57:1003-1012).
Mutagenesis by genetic engineering has been used extensively in order to elucidate the structural organization of functional domains of recombinant proteins. Several different approaches have been described in the literature for carrying out deletion or substitution mutagenesis. The most successful appear to be alanine scanning mutagenesis [Cunningham and Wells 1989, Science 244: 1081-1085] and homolog-scanning mutagenesis [Cunningham et al., 1989, Science 243:1330-1336]. These approaches helped identify the receptor binding domains of growth hormone and create hybrid proteins with altered binding properties to their cognate receptors.
To better understand the physical, biochemical and pharmacological properties of rHCNTF, applicant undertook rational mutagenesis of the human and rat CNTF genes based on the different biological and physical properties of their corresponding recombinant proteins (See Masiakowski, P., et al., 1991, J. Neurochem., 57:1003-1012). Applicant has found that the nature of the amino acid at position 63 could greatly enhance the affinity of human CNTF for sCNTFRxcex1 and its biological potency in vitro (Panayotatos, N., et al. , J. Biol. Chem., 1993, 268:19000-19003; Panayotatos, N., et al., Biochemistry, 1994, 33: 5813-5818.
As described in copending U.S. patent application Ser. No. 07/570,651 filed Aug. 20, 1990, entitled xe2x80x9cCiliary Neurotrophic Factorxe2x80x9d and International Publication Number WO 91/04316 published Apr. 4, 1991 which are incorporated by reference in their entireties herein, one of the uses of CNTF contemplated by applicants was the use of CNTF for the treatment of Huntington""s disease (Huntington""s chorea). Huntington""s disease (HD) is an hereditary degenerative disorder of the central nervous system. The pathology underlying HD is progressive, relentless degeneration of the basal ganglia, structures deep inside the brain which are responsible for aspects of the integration of voluntary motor and cognitive activity. The onset of symptoms in HD is generally in adulthood, between the ages of 20 and 40. The characteristic manifestations of the disease are chorea and other involuntary movements, dementia, and psychiatric symptoms. Choreic movements consist of brief, involuntary, fluid movements, predominantly affecting the distal extremities. Patients often tend to xe2x80x9ccover upxe2x80x9d these movements by blending them into voluntary acts. HD patients also, however, display a variety of other neurological abnormalities including dystonia (sustained, abnormal posturing), tics (xe2x80x9chabit spasmsxe2x80x9d), ataxia (incoordination) and dysarthria (slurred speech). The dementia of HD is characterized as the prototypical xe2x80x9csubcorticalxe2x80x9d dementia. Manifestations of dementia in HD include slowness of mentation and difficulty in concentration and in sequencing tasks. Behavioral disturbances in HD patients are varied, and can include personality changes such as apathy and withdrawal; agitation, impulsiveness, paranoia, depression, aggressive behavior, delusions, psychosis, etc. The relentless motor, cognitive and behavioral decline results in social and functional incapacity and, ultimately death.
HD is inherited as an autosomal dominant trait. Its prevalence in the U.S. population is estimated to be 5 to 10 per 100,000 individuals, yielding a total prevalence of 25,000 in the US population. However, due to the late onset of symptoms, there are a number of xe2x80x9cat-riskxe2x80x9d, asymptomatic individuals in the population as well. The prevalence of asymptomatic, at-risk patients carrying the HD gene is perhaps twice that of the symptomatic patients (W. Koroshetz and N. Wexler, personal communication). Thus, the total HD patient population eligible to receive a new therapy is about 75,000.
The gene currently believed to be responsible for the pathogenesis of HD is located at the telomeric end of the short arm of Chromosome 4. This gene codes for a structurally novel protein of unknown function, and the relationship of the gene product to the pathogenesis of HD remains uncertain at the present time.
The principal anatomical lesion in HD consists of loss of the so-called xe2x80x9cmedium spinyxe2x80x9d neurons of the caudate nucleus and putamen (collectively known as the striatum in rodents). These neurons comprise the projection system whereby the caudate/putamen projects to its output nuclei elsewhere in the basal ganglia of the brain. The principal neurotransmitter utilized by the medium spiny neurons is gamma-aminobutyric acid (GABA), although many also contain neuropeptides such as enkephalins and substance P. It is clear, however, that in HD interneurons which do not utilize GABA as their neurotransmitter, containing instead either acetylcholine or the neuropeptides somatostatin or neuropeptide Y, are relatively undamaged in HD.
Pathological and neurochemical changes which mimic those seen in HD can be mimicked by infusion of glutamatergic agonist drugs into the striatum. Infusion of quinolinic acid under appropriate conditions produces selective depletion of medium sized intrinsic striatal neurons which utilize gamma-aminobutyric acid (GABA) as their neurotransmitter, without affecting the large, cholinergic interneurons.
There have been no successful clinical trials of either symptomatic or neuroprotective treatments in HD. However, useful, validated rating instruments and neuroimaging techniques exist which are capable of monitoring disease progress and patient function.
The CNTF receptor complex contains 3 proteins: a specificity determining a component that directly binds to CNTF, as well as 2 signal transducing b components (LIFR b and gp130) that cannot bind CNTF on their own, but are required to initiate signaling in response to CNTF. The b component of the CNTFR complex is more widely distributed throughout the body than the a component. The 3 components of the CNTFR complex are normally unassociated on the cell surface; CNTF induces the stepwise assembly of a complete receptor complex by first binding to CNTFR a, then engaging gp130, and finally recruiting LIFR b. When this final step in receptor assembly occurs (heterodimerization of the b components), intracellular signaling is initiated by activating non-receptor tyrosine kinases (JAK kinases) associated with the b components. JAK kinases respond by phosphorylating each other and also tyrosine residues on the receptor cytoplasmic domains, creating phosphotyrosine docking sites for the Src homology 2 domains of STAT proteins. After their phosphorylation, bound STAT proteins dissociate from the receptor, dimerize, and translocate to the nucleus where they bind DNA and activate transcription (reviews: Frank, D. and Greenberg, M. (1996) Perspectives on developmental neurobiology 4: 3-18; Stahl, N. and Yancopoulos, G. (1997) Growth factors and cytokines in health and disease 2B, 777-809). Axokine(trademark) (hCNTF C17A, Q63R, xcex9415) (Ax-15) is a mutant CNTF molecule with improved physical and chemical properties, which retains the ability to interact with and activate the CNTF receptor. (Panayotatos, N., et al. (1993) J. Biol. Chem. 268: 19000-19003).
Leptin, the product of the ob gene, is secreted by adipocytes and functions as a peripheral signal to the brain to regulate food intake and energy metabolism (Zhang, Y., et al. (1994) Nature 372: 425-431). Interestingly, leptin receptor (OB-R), a single membrane-spanning receptor has considerable sequence similarities to gp130 (Tartaglia, L., et al. (1995) Cell 83: 1263-1271), and like CNTF, leptin signals through the JAK/STAT pathway (Baumann, H., et al. (1996) Proc. Natl. Acad. Sci. USA 93: 8374-8378; Ghilardi, N., et al. (1996) Proc. Natl. Acad. Sci. USA 93: 6231-6235). Systemic administration of both CNTF and leptin resulted in induction of tis-11 (Gloaguen, I., et al. (1997) Proc. Natl. Acad. Sci. USA 94: 6456-6461) and STAT3 (Vaisse, C., et al. (1996) Nature Gen. 14: 95-97) in the hypothalamic satiety center, indicating their roles in the regulation of body weight and feeding behavior. Indeed, adminstration of CNTF to humans reduced food intake and resulted in weight loss (Group, A. C. T. S. (1996) Neurology 46:1244-1249.).
An object of the present invention is to provide novel CNTF-related neurotrophic factors for the treatment of diseases or disorders including, but not limited to, diabetes and obesity. In a preferred embodiment, CNTF and related molecules are utilized for the treatment of non-insulin dependent diabetes mellitus.
A further object of the present invention is to provide a method for identifying CNTF-related factors, other than those specifically described herein, that have improved therapeutic properties.
These and other objects are achieved in accordance with the invention, whereby amino acid substitutions in human CNTF protein enhance its therapeutic properties. In one embodiment, alterations in electrophoretic mobility are used to initially screen potentially useful modified CNTF proteins.
In a preferred embodiment, the amino acid glutamine in position 63 of human CNTF is replaced with arginine (referred to as 63Qxe2x86x92R) or another amino acid which results in a modified CNTF molecule with improved biological activity. In further embodiments, rHCNTF variants combine the 63Qxe2x86x92R mutation with three other novel features:
1) Deletion of the last 13 amino acid residues (referred to as xcex94C13) to confer greater solubility to rHCNTF without impairing its activity;
2) Substitution of the unique cysteine residue at position 17, which results in stabilization of rHCNTF in physiological buffer, at physiological pH and temperature conditions without affecting its activity; or
3) Substitution of amino acid residue 64W, which alters the biological activity of rHCNTF in vitro and which results in a 7-fold improvement of its therapeutic index in vivo.
In another preferred embodiment, a molecule designated RG297 (rHCNTF,17CA63QRxcex94C13) combines a 63Qxe2x86x92R substitution (which confers greater biological potency) with a deletion of the terminal 13 amino acid residues (which confers greater solubility under physiological conditions) and a 17CA substitution (which confers, stability, particularly under physiological conditions at 37xc2x0 C.) and shows a 2-3 fold better therapeutic index than rHCNTF in an animal model.
In another preferred embodiment, a molecule designated RG242 is described that carries the double substitution 63QR64WA which results in a different spectrum of biological potency and a 7-fold higher therapeutic index.
In another preferred embodiment, a molecule designated RG290 is described that carries the double substitution 63QRxcex94C13 which confers greater solubility under physiological conditions.